Investigations into the roles of Histamine Receptor H1 and extracellular vesicles in ovarian cancer cell behaviour

Ovarian cancer (OC) diagnosis usually occurs very late, when metastatic spread has already started and patient’s survival is very low. One of the many soluble factors that can promote cancer metastasis is histamine, a compound involved in a plethora of physiological and pathological processes, including cancer. Indeed, activation of histamine receptor H1 (HRH1) by histamine stimulates growth of OC cells in vitro and promotes the release of extracellular vesicles (EVs) in different cell lines. EVs are heterogeneous small vesicles involved in intracellular communication, which also modulate various steps of the metastatic process. The main hypothesis of this thesis is that HRH1 mediates several cancerous behaviours associated with OC metastatic spread, by regulating EV release. The specific aims were 1) to analyse the correlation of histamine receptors gene expression with invasion and migration rates of OC cell lines in vitro and with tumour stage of OC clinical samples; 2) to study the involvement of HRH1 in different cellular behaviours associated with cancer metastasis; 3) to investigate the involvement of HRH1 in EV release and how they affect OC cell invasion in vitro. The results show that the level of HRH1 mRNA positively correlates with in vitro migration and invasion rate of OC cells lines and higher expression was found in stage IV of OC clinical samples compared to stage II/III. Low HRH1 correlates with increased disease free survival, although no correlation was found with overall survival. HRH1 expression was modulated in three OC cell lines (SKOV3, OVCAR3 and OVCAR5), via siRNA transfection, chemical activation (via histamine) or inhibition (via chlorpheniramine) and its effect on cellular behaviour was evaluated through different in vitro assays. Results showed that HRH1: 1) does not modulate gene expression of EMT-related genes; 2) does not influence adhesion of OC cells to endothelial cells; 3) reduces cell invasion through a Matrigel®️ layer and 4) reduces their movement from the edge of a simulated ‘wound’. Histamine increases the number of EVs released from SKOV3 cells and their ability to degrade a collagen substrate but did not modify EV protein contents compared to control EVs. EVs were able to rescue the reduction of invasion caused by HRH1 knockdown, while histamine failed to rescue the invasion of cells knocked down for Rab27a (a major regulator of EV production), suggesting a potential interplay between HRH1 and Rab27a in modulating EV release and cell invasion. Overall, these results suggest that HRH1, via modulation of EV biogenesis, can impair OC cells invasion and migration in vitro

Extracellular vesicles:Emerging modulators of cancer drug resistance

Extracellular vesicles (EVs) have recently emerged as crucial modulators of cancer drug resistance. Indeed, it has been shown that they can directly sequester anti-tumor drugs, decreasing their effective concentration at target sites. Moreover, they facilitate the horizontal transfer of specific bioactive cargoes able to regulate proliferative, apoptotic, and stemness programs in recipient cells, potentially conferring a resistant phenotype to drug-sensitive cancer cells. Finally, EVs can mediate the communication between the tumor and both stromal and immune cells within the microenvironment, promoting treatment escape. In this context, clarifying the EV-driven resistance mechanisms might improve not only tumor diagnosis and prognosis but also therapeutic outcomes. Detailed cellular and molecular events occurring during the development of EV-mediated cancer drug resistance are described in this review article

Detecting ovarian cancer using extracellular vesicles: Progress and possibilities

Ovarian cancer (OC) is the deadliest gynecological malignancy. Most patients are diagnosed when they are already in the later stages of the disease. Earlier detection of OC dramatically improves the overall survival, but this is rarely achieved as there is a lack of clinically implemented biomarkers of early disease. Extracellular vesicles (EVs) are small cell-derived vesicles that have been extensively studied in recent years. They contribute to various aspects of cancer pathology, including tumour growth, angiogenesis and metastasis. EVs are released from all cell types and the macromolecular cargo they carry reflects the content of the cells from which they were derived. Cancer cells release EVs with altered cargo into biofluids, and so they represent an excellent potential source of novel biomarkers for the disease. In this review we describe the latest developments in EVs as potential biomarkers for earlier detection of OC. The field is still relatively young, but a number of studies have shown that EVs and the cargo they carry, including miRNAs and proteins, can be used to detect OC. They could also give insight into the stage of the disease and predict the likely therapeutic outcome. There remain a number of challenges to the use of EVs as biomarkers, but through ongoing research and innovation in this exciting field there is great potential for the development of diagnostic assays in the clinic that could improve patient outcome

A practical toolkit to study aspects of the metastatic cascade in vitro

While metastasis – the spread of cancer from the primary location to distant sites in the body – remains the principle cause of cancer death, it is incompletely understood. It is a complex process, requiring the metastatically successful cancer cell to negotiate a formidable series of interconnected steps, which are described in this paper. For each step, we review the range of in vitro assays that may be used to study them. We also provide a range of detailed, step-by-step protocols that can be undertaken in most modestly-equipped laboratories, including methods for converting qualitative observations into quantitative data for analysis. Assays include: (1) a gelatin degradation assay to study the ability of endothelial cells to degrade extracellular matrix during tumour angiogenesis; (2) the morphological characterisation of cells undergoing epithelial-mesenchymal transition (EMT) as they acquire motility; (3) a ‘scratch’ or ‘wound-healing’ assay to study cancer cell migration; (4) a transwell assay to study cancer cell invasion through extracellular matrix; and (5) a static adhesion assay to examine cancer cell interactions with, and adhesion to, endothelial monolayers. This toolkit of protocols will enable researchers who are interested in metastasis to begin to focus on defined aspects of the process. It is only by further understanding this complex, fascinating and clinically relevant series of events that we may ultimately devise ways of better treating, or even preventing, cancer metastasis. The assays may also be of more broad interest to researchers interested in studying aspects of cellular behaviour in relation to other developmental and disease processes

VATS Pleurectomy Decortication Is a Reasonable Alternative for Higher Risk Patients in the Management of Malignant Pleural Mesothelioma: An Analysis of Short-Term Outcomes

Surgery is a mainstay of treatment allowing for debulking of tumor and expansion of the lung for improvement in median survival and quality of life for patients with malignant pleural mesothelioma (MPM). Although optimal surgical technique remains open for debate—extrapleural pneumonectomy (EPP) vs. pleurectomy/decortication (P/D)—minimally invasive surgery (VATS-P/D) remains underutilized in the management of MPM. We examined whether VATS-P/D is a feasible alternative to EPP and P/D. We evaluated the New York Statewide Planning and Research Cooperative System (SPARCS) from 2007–2017 to assess the short-term complications of EPP vs. P/D, including a subanalysis of open P/D vs. VATS-P/D. There were 331 patients with open surgery; 269 with P/D and 62 with EPP. There were 384 patients with P/D; 269 were open and 115 VATS. Rates of any complication were similar between EPP and P/D patients, but EPP had significantly higher rates of cardiovascular complications. After adjusting for confounders, those with a VATS approach were less likely to have any complication, compared to an open approach and significantly less likely to have a pulmonary complication. VATS-P/D remains a viable alternative to radical surgery in MPM patients allowing for improved short-term outcomes

Extracellular Vesicles: Emerging Modulators of Cancer Drug Resistance

Extracellular vesicles (EVs) have recently emerged as crucial modulators of cancer drug resistance. Indeed, it has been shown that they can directly sequester anti-tumor drugs, decreasing their effective concentration at target sites. Moreover, they facilitate the horizontal transfer of specific bioactive cargoes able to regulate proliferative, apoptotic, and stemness programs in recipient cells, potentially conferring a resistant phenotype to drug-sensitive cancer cells. Finally, EVs can mediate the communication between the tumor and both stromal and immune cells within the microenvironment, promoting treatment escape. In this context, clarifying the EV-driven resistance mechanisms might improve not only tumor diagnosis and prognosis but also therapeutic outcomes. Detailed cellular and molecular events occurring during the development of EV-mediated cancer drug resistance are described in this review article

Adipocyte-Derived Extracellular Vesicles Promote Prostate Cancer Cell Aggressiveness by Enabling Multiple Phenotypic and Metabolic Changes

Background: In recent decades, obesity has widely emerged as an important risk factor for prostate cancer (PCa). Adipose tissue and PCa cells have been shown to orchestrate a complex interaction network to support tumor growth and evolution; nonetheless, the study of this communication has only been focused on soluble factors, although increasing evidence highlights the key role of extracellular vesicles (EVs) in the modulation of tumor progression. Methods and Results: In the present study, we found that EVs derived from 3T3-L1 adipocytes could affect PC3 and DU145 PCa cell traits, inducing increased proliferation, migration and invasion. Furthermore, conditioning of both PCa cell lines with adipocyte-released EVs resulted in lower sensitivity to docetaxel, with reduced phosphatidylserine externalization and decreased caspase 3 and PARP cleavage. In particular, these alterations were paralleled by an Akt/HIF-1α axis-related Warburg effect, characterized by enhanced glucose consumption, lactate release and ATP production. Conclusions: Collectively, these findings demonstrate that EV-mediated crosstalk exists between adipocytes and PCa, driving tumor aggressiveness

Myasthenia Gravis Treatment: From Old Drugs to Innovative Therapies with a Glimpse into the Future

: Myasthenia gravis (MG) is a rare autoimmune disease that causes debilitating muscle weakness due to impaired neuromuscular transmission. Since most (about 80-90%) MG patients present autoantibodies against the acetylcholine receptor, standard medical therapy consists of symptomatic treatment with acetylcholinesterase inhibitors (e.g., pyridostigmine). In addition, considering the autoimmune basis of MG, standard therapy includes immunomodulating agents, such as corticosteroids, azathioprine, cyclosporine A, and cyclophosphamide. New strategies have been proposed for the treatment of MG and include complement blockade (i.e., eculizumab, ravulizumab, and zilucoplan) and neonatal Fc receptor antagonism (i.e., efgartigimod and rozanolixizumab). The aim of this review is to provide a detailed overview of the pre- and post-marketing evidence on the five pharmacological treatments most recently approved for the treatment of MG, by identifying both preclinical and clinical studies registered in clinicaltrials.gov. A description of the molecules currently under evaluation for the treatment of MG is also provided